Cancers have altered transcriptional programs that lead to deregulation of normal cellular functions and support the cancer cell characteristics. A prerequisite for the cancer cell is an increase in its ribosynthetic activity to support the increaed needs for protein synthesis. These processes are governed by RNA polymerase I (Pol I), which is highly responsive to external stimuli and commonly deregulated in cancer. Yet, attempts to exploit Pol I as a clinically relevant target have been limited and intervention modalities are scarce. We have recently discovered a unique small molecule pyridoquinazolinecarboxamide (BMH-21) that causes Pol I transcription blocks, is broadly effective against many cancer cell types and is well tolerated in normal cells and in mouse. These findings for the first time facilitate approaches analyzing the intersection of Pol I targeting in the context of cancer therapeutic targets, genetic features and altered pathways. We hypothesize that the Pol I inhibitory activity elicited by BMH-21, and its anticancer activity can be amplified through combinatory therapies or tumor-specific pathway defects. The rationale for this exploratory project is that there is no existing data how inhibition of Pol I intersects with other cancer cell intrinsic deficiencies or targetable events. The goal of this exploratory grant application is to build a sound rational basis towards the efforts to identify factors that sensitize cancer cells to Pol I inhibition. Towards this goal, chemogenomic synthetic lethality screens will be conducted that exploit cancer cell lethality that arises from inhibition of Pol I, cancer cell defects or therapeutically actionable events. Aim 1 will use an RNAi screen to investigate genetic pathways that sensitize cancer cells to Pol I transcription blocks by BMH-21. Aim 2 determines synthetic lethality of Pol I inhibition based on the pathway defects. It implements testing of combinatory activities of known drugs and experimental molecules with BMH-21, and assessment of the most promising combinations in mouse tumor xenograft models. The chemogenomic profiling will define actionable events increasing the sensitivity of cancers to Pol I-based therapies. Identification of genes and their associated pathways that sensitize cancer cells to Pol I inhibition will guide the linking of cancer genetic events predisposing to Pol I inhibition and which could be exploited clinically in future. This project will lead to further mechanistic and definitive studies that analyze the molecular underpinnings of the combinatory activities. In all, these studies provide novel insight into the intersection of Pol I and cancer deregulated pathways and support the development of rational, improved cancer therapies.